Glycosylation of peptide chains is one of the most important processes of post-translational modification. Glycoproteins containing sugar chains attached to peptide chains are involved in various life phenomena. It is believed that, in vivo, intercellular signal transduction, molecular recognition, etc. are controlled by precisely recognizing slight structural differences of sugar chains Therefore, structural analysis of glycoproteins or glycopeptides is expected to make a major contribution to elucidation of life phenomena, drug discovery, biomarker development, etc.
An N-linked sugar chain attached to an asparagine residue of a protein has at least one branch, and often has sialic acid at its non-reducing end. Sialic acid at the non-reducing end of a sugar chain is directly involved in molecular recognition, and therefore the analytical determination of the presence or absence of sialic acid (the number of sialic acid residues) and the linkage type of sialic acid is important in structural analysis of glycoproteins or glycopeptides.
Sialic acid has a negative charge, and is unstable and is therefore easily decomposed or detached from sugar chains. For this reason, some analytical methods have been proposed in which sialic acid is stabilized by chemical modification before analysis. For example, Patent Document 1 discloses a method in which the reducing end of a free sugar chain is immobilized on a solid-phase carrier, and the carboxy group of sialic acid at the non-reducing end of the sugar chain is methylamidated using PyAOP as a condensation agent and methylamine hydrochloride as a nucleophile. Further, Patent Document 1 discloses an example in which a sample after modification by methylamidation is subjected to mass spectrometry to perform quantitative determination and structural analysis of the sugar chain.
Patent Document 2 states that the detachment of sialic acid during mass spectrometry (ionization) is prevented by modifying (or removing) all the carboxy groups present in a glycopeptide by reaction in the presence of a dehydration-condensation agent such as a phosphonium salt. Further, Patent Document 2 states that the branching structure of sialic acid-containing sugar chain of a glycopeptide can be analyzed by subjecting a sample after modification to multi-stage mass spectrometry.
Mass spectrometry is an effective analysis method for structural analysis of sugar chains. As described above, the presence or absence of sialic acid and the branching structure of a sugar chain can also be analytically determined by structurally stabilizing sialic acid at the non-reducing end by modification. On the other hand, the methods disclosed in Patent Document 1 and Patent Document 2 cannot identify the linkage type of sialic acid, because methylamidation is performed independently of the linkage type of sialic acid.
As the linkage types of sialic acid to the non-reducing end of a sugar chain, there are mainly α2,3- and α2,6-linked isomers. It is known that in vivo, a difference in the linkage type of sialic acid is involved in various life phenomena. For example, it is known that the linkage type of sialic acid changes with canceration. Therefore, identifying a difference in the linkage type of sialic acid is attracting attention as a biomarker or in quality control of biopharmaceuticals, etc.
In order to identify the linkage type of sialic acid by mass spectrometry, linkage type-specific modification needs to be performed so that sialic acid has a mass different depending on its linkage type. For example, Patent Document 3 proposes a method in which methylesterification of sialic acid is performed using 1-methyl-3-p-tolyltriazene (MTT), and then an acidic condition is created. Patent Document 3 states that this method can discriminate between α2,3-linked sialic acid and α2,6-linked sialic acid, because only α2,3-linked sialic acid is selectively demethylated under an acidic condition.
Further, a method for identifying the linkage type of sialic acid by mass spectrometry has also been proposed which utilizes the fact that α2,3-linked sialic acid easily form a lactone ring by intramolecular dehydration condensation in the presence of a dehydration-condensation agent. For example, Non-Patent Document 1 and Non-Patent Document 2 disclose that when a sugar chain sample and a dehydration-condensation agent are present in methanol or ethanol, α2,6-linked sialic acid is preferentially esterified, and α2,3-linked sialic acid preferentially forms a lactone ring by intramolecular dehydration. Non-Patent Document 3 discloses a method in which a sugar chain sample is reacted with ammonium chloride to lactonize and amidate α2,3-linked sialic acid and α2,6-linked sialic acid, respectively and then they are completely methylated. When these modification methods are used, a modified compound of α2,3-linked sialic acid and a modified compound of α2,6-linked sialic acid have different masses, which makes it possible to identify the linkage type of sialic acid by mass spectrometry.